Attachment of Entamoeba histolytica to glass in a defined maintenance medium: specific requirement for cysteine and ascorbic acid

Cysteine and ascorbic acid were previously shown to be required by Entamoeba histolytica trophozoites for attachment to glass, elongation, and ameboid movement as well as for short-term (12-24 h) survival in a balanced salt solution containing bovine serum albumin and a vitamin solution (Maintenance Medium 1). If the only function of cysteine and ascorbate was to decrease the redox potential, other reducing agents should be effective. However, the requirement for cysteine in the presence of ascorbic acid was highly specific. Equally effective were D- and L-cysteine; however, of many other compounds tested, only thioglycolic acid, ascorbic acid, or L-cystine (in decreasing order) were somewhat active. Under N2 atmosphere, cysteine and ascorbic acid were still required, although their concentrations could be halved. The ability to attach in the maintenance medium was irreversibly lost after only 5 min of cysteine-ascorbic acid deprivation; however, there was no decrease in viability when the amebae were transferred to growth medium within 30 min. Cysteine thiol groups in the medium were oxidized rapidly regardless of the concentration of ascorbic acid or the presence of amebae; however, ascorbic acid prolonged attachment of amebae.     

Interactions of Giardia lamblia with human intestinal mucus: enhancement of trophozoite attachment to glass

Giardia lamblia trophozoites frequently are associated with mucus in vivo. We investigated the effects of human intestinal mucus on parasite attachment and survival in vitro. All samples of mucus from the duodenum and ileum (from four humans and two rabbits) enhanced attachment at 100 micrograms/ml. Attachment increased with mucus concentrations from 1 to 1000 micrograms/ml but declined toward the unstimulated level at concentrations above 1000 micrograms/ml. Mucus from the small intestine also promoted the survival of the parasites during the 2-h incubation. In contrast, colonic mucus promoted survival, but inhibited attachment. Fractionation of mucus from the human small intestine by cesium chloride equilibrium density gradient ultracentrifugation revealed that both attachment- and survival-promoting activities were in the low density, protein-rich fraction. The high density fractions containing the mucins were devoid of activity. Thus, a non-mucin fraction of mucus from the human small intestine may promote colonization by G. lamblia.     

Sorting of cyst wall proteins to a regulated secretory pathway during differentiation of the primitive eukaryote, Giardia lamblia

Giardia lamblia, which belongs to the earliest identified lineage to diverge from the eukaryotic line of descent, is one of many protists reported to lack a Golgi apparatus. Our recent finding of a developmentally regulated secretory pathway in G. lamblia makes it an ideal organism with which to test the hypothesis that the Golgi may be more readily demonstrated in actively secreting cells. These ultrastructural studies now show that a regulated pathway of transport and secretion of cyst wall antigens via a novel class of large, osmiophilic secretory vesicles, the encystation-specific vesicles (ESV), is assembled during encystation of G. lamblia. Early in encystation, cyst antigens are localized in simple Golgi membrane stacks and concentrated within enlarged Golgi cisternae which appear to be precursors of ESV. This would represent an unusual mechanism of secretory vesicle biogenesis. Later in differentiation, cyst antigens are localized within ESV, which transport them to the plasma membrane and release them by exocytosis to the nascent cell wall. ESV are not observed after completion of the cyst wall. In contrast to the regulated transport of cyst wall proteins, we demonstrate a distinct constitutive lysosomal pathway. During encystation, acid phosphatase activity is localized in endoplasmic reticulum, Golgi, and small constitutive peripheral vacuoles which function as lysosomes. However, acid phosphatase activity is not detectable in ESV. These studies show that G. lamblia, an early eukaryote, is capable of carrying out Golgi-mediated sorting of proteins to distinct regulated secretory and constitutive lysosomal pathways.     

Reproductive and neonatal outcomes in captive bred baboons (Papio hamadryas)

Abstract: Baboons are widely used in biomedical research. Although it is widely held that Papio hamadryas breed well in captivity, each established colony has a different reproductive success often hypothesised to be due to husbandry practices. The National Baboon Colony in Australia is a unique colony that houses Papio hamadryas to mimic that structure seen in the wild. In this article; we have analysed their reproductive parameters and neonatal outcomes. The success of the colony husbandry practices was demonstrated by lack of maternal mortality, low foetal morbidity, and known maternal and paternal linage.     

Reduction of REM sleep by a tryptophan-free amino acid diet.

The acute administration of a tryptophan-free amino acid diet to rats has previously been reported to produce a marked reduction in brain serotonin concentrations. The present study examined the effects of such a diet on electroencephalographic sleep measures. There was a decrease in REM sleep and a small increase in nonREM sleep, with no change in total sleep time. In view of these and other observations, the hypothesis that the serotonergic system plays an important role in the maintenance of nonREM sleep should be carefully reevaluated.     

Entamoeba histolytica and Giardia lamblia: Growth responses to reducing agents

The growth responses of Entamoeba histolytica strains HM-1:IMSS and HK-9 to a variety of reducing agents were tested for one subculture in TYI-S-33 medium, prepared with no cysteine or ascorbic acid. Amoebae did not grow in this medium. Addition of l-ascorbic acid, d- or l-cysteine, or l-cystine each permitted the maximum growth observed. Dithiothreitol supported 68% maximum growth of HK-9 amoebae, but only 12% of HM-1. In contrast, growth of both strains was greatly diminished (0–13% growth) with 11 other compounds tested including glutathione, thiomalic acid, thioglycolic acid, and methionine. The growth responses of Giardia lamblia were similarly tested in TYI-S-33, as well as in TP-S-1 media. If l-cysteine was omitted from either medium, trophozoites did not grow, and eventually lysed. In TYI-S-33 medium, the requirement for l-cysteine was specific, whereas in TP-S-1 medium, other sulfhydryl compounds were partially effective and lower concentrations of l-cysteine satisfied the requirement. Ascorbic acid or l-cystine alone was totally ineffective; however, in combination, 30 to 60% of maximum growth was achieved. Once added to either medium, cysteine was rapidly oxidized. Amino acid analysis of the growth media revealed that the broth components of TP-S-1 medium contained 2.8 mM and TYI-S-33 broth 2.1 mM endogenous levels of cysteine (or half-cystine), with an additional 3 mM contributed by 10% serum.     

MASS FRAGMENTOGRAPHIC DETERMINATION OF SOME ACIDIC AND ALCOHOLIC METABOLITES OF BIOGENIC AMINES IN THE RAT BRAIN

—A mass fragmentographic procedure is described for the simultaneous quantification of a number of deaminated metabolites derived from tyramine, octopamine, dopamine, and norepinephrine. With this method, several of the metabolites were measured in normal rat brain. The results support the central nervous system origin of tyramine, octopamine and their metabolites. The concentration of the dopamine metabolite, homovanillic acid, in the rat brain was found to be about 15% higher than that of dihydroxyphenylacetic acid. As for the metabolites of norepinephrine, vanilmandelic acid concentration was found to be about 5% that of 3-methoxy-4-hydroxyphenylglycol. The possible role of vanilmandelic acid in the CNS metabolism of norephrine is discussed.     

Mining the Giardia lamblia genome for new cyst wall proteins

The Giardia lamblia cyst wall (CW), which is required for survival outside the host and infection, is a primitive extracellular matrix. Because of the importance of the CW, we queried the Giardia Genome Project Database with the coding sequences of the only two known CW proteins, which are cysteine-rich and contain leucine-rich repeats (LRRs). We identified five new LRR-containing proteins, of which only one (CWP3) is up-regulated during encystation and incorporated into the cyst wall. Sequence comparison with CWP1 and -2 revealed conservation within the LRRs and the 44-amino-acid N-flanking region, although CWP3 is more divergent. Interestingly, all 14 cysteine residues of CWP3 are positionally conserved with CWP1 and -2. During encystation, C-terminal epitope-tagged CWP3 was transported to the wall of water-resistant cysts via the novel regulated secretory pathway in encystation-secretory vesicles (ESVs). Deletion analysis revealed that the four LRRs are each essential to target CWP3 to the ESVs and cyst wall. In a deletion of the most C-terminal region, fewer ESVs were stained in encysting cells, and there was no staining in cysts. In contrast, deletion of the 44 amino acids between the signal sequence and the LRRs or the region just C-terminal to the LRRs only decreased the number of cells with CWP3 targeting to ESVs and cyst wall by approximately 50%. Our studies indicate that virtually every portion of the CWP3 protein is needed for efficient targeting to the regulated secretory pathway and incorporation into the cyst wall. Further, these data demonstrate the power of genomics in combination with rigorous functional analyses to verify annotation.